1. Field of the Invention
The present invention relates to a method and a reagent for measuring cell counts or the presence of cells with high sensitivity by measuring intracellular ATP. In particular, the present invention relates to a method and a reagent for measuring cells (particularly viable cells) in a sample.
2. Background Art
Methods for measuring cell counts are employed in a broad range of fields, including the pharmaceutical, medical, food, brewing, and water treatment fields. In particular, in various fields such as pharmaceutical manufacturing, cosmetics manufacturing, clinical medicine, and basic biochemical fields, the presence or the absence of cells in samples is determined and cell counts are measured for the quality control of samples. For example, it is essential in the pharmaceutical manufacturing field to control microorganisms (bacteria and fungi) contained in pharmaceutical raw materials, pharmaceutical intermediates, final pharmaceutical products, and pharmaceutical water according to the Japanese Pharmacopoeia as standards stipulated by the Ministry of Health, Labour and Welfare. The number of microorganisms is measured daily.
Bacterial or fungal counts are mainly measured by a culture method as specified in the Japanese Pharmacopoeia. Such culture method involves bringing a sample into contact with an agar plate medium for culture, and then quantifying the number of colonies (CFU: Colony Forming Unit) as the number of microorganisms (microbial count) in the sample, with the use of the fact that one microorganism forms one colony as a result of culture.
Culture methods in the pharmaceutical industry have a problem that these methods require about 1 week for culture. Manufacturing is delayed at the intermediate production and final product shipping stages to wait for measurement results. Thus, such culture methods create temporal and economic burdens on the industry. In addition, culture methods necessitate choosing a specific medium types for each microbial species. A medium having a nutrient appropriate for the colony formation of each microorganism must be selected (Non-patent Document 1). For example, a standard agar medium is selected for general aerobic bacteria, R2A medium is selected for oligotrophic bacteria, a thioglycol medium is selected for anaerobic bacteria, and PDA medium is selected for fungi.
Specifically, aerobic bacteria are cultured in a standard agar medium at temperatures ranging from 30° C. to 35° C. for 48 to 72 hours, anaerobic bacteria are cultured in a thioglycolic acid medium at temperatures ranging from 30° C. to 35° C. for 5 or more days, oligotrophic bacteria are cultured in R2A medium at temperatures ranging from 20° C. to 25° C. (or 30° C. to 35° C.) for 4 to 7 days, and fungi are cultured in PDA medium at temperatures ranging from 20° C. to 25° C. for 5 or more days, for example. Hence, a plurality of protocols varying in medium, temperature, time for culture, and others appropriate for the colony formation of each microbial species are required. Accordingly, a single sample may need to be subdivided into a plurality of samples, or a plurality of samples may be required. Another problem is that a maximum of 7 days of culture is required for colony formation. Therefore, it has been difficult to measure the microbial counts of all microbial species using only one sample. Thus, measurement using a plurality of samples and a plurality of media has been essential.
An ATP (Adenosine TriPhosphate) luminescence method has been known as a method to rapidly measure cell counts with a shortened time of measurement. ATP to be measured is an organic compound that is contained within cells of all organisms and serves as an energy source required by cells' biological activity. The ATP luminescence method involves, with the use of luciferase and luciferin that chemically react with ATP to emit luminescence, measuring luminescence generated by a reaction of intracellular ATP with luciferase and luciferin, and thus estimating the cell count based on the luminescence level.
It has been generally known that when the ATP luminescence method is applied for cell counting, accurate viable cell counts cannot be estimated by directly measuring a sample, since ATP from viable cells (living microorganisms or live cells), ATP from dead cells (dead microorganisms), and free ATP are mixed within the single sample. To address this problem, an ATP elimination method using adenosine-phosphate deaminase, apyrase, and others in combination has been known (Patent Document 1).
According to a conventional intracellular ATP measurement method 101 (for measuring ATP within viable cells) (FIG. 1), a sample 102 containing viable-cell-derived ATP, dead-cell-derived ATP, and free ATP is subjected to three stages: (1) extracellular ATP removal 103 (for removing ATP outside of viable cells); (2) intracellular ATP extraction 104 (for extracting ATP within viable cells); and (3) the luminescent reaction of viable-cell-derived ATP with a luminescent reagent (e.g., luciferase and luciferin) and luminescence measurement 105. Therefore, the viable cell count is estimated based on luminescence resulting from the luminescent reaction of ATP within viable cells with the luminescent reagents.
The states of viable-cell-derived ATP 122, dead-cell-derived ATP, free ATP 123, and ATPase 124 in each step of the conventional intracellular ATP measurement method 101 (for measuring ATP within viable cells) and an ATP luminescence method 121 are as explained below.
In the extracellular ATP removal step 103 (for removing ATP outside of viable cells), dead-cell-derived ATP and free ATP 123 are mainly removed. In the removal method, ATPase 124 (e.g., apyrase) is added to a sample (106). Cell walls or cell membranes in dead cells are disrupted, therefore, ATP within dead cells and free ATP 123 are degraded by the activity 125 of ATPase 124 (126). On the other hand, viable cells 122 are living 127, and thus ATP within viable cells is isolated from ATPase 124 because of viable cells' walls or membranes (Patent Document 2). Patent Document 2 discloses that ATP within viable cells such as Escherichia coli, Lactobacillus brevis, Staphyllococcus aureus, and Bacillus subtilis can be increased following culture in standard media by adding glucose. Moreover, Patent Document 3 discloses that spore germination can be induced by adding glucose and alanine to spores.
In the intracellular ATP extraction step 104 (for extracting ATP within viable cells), “disruption of viable cells' walls” and “inactivation of ATPase” are performed. An ATP extraction method is performed by adding an ATP extraction solution 107 of trichloroacetic acid, benzalkonium chloride, methanol or the like (Patent Document 2). In the ATP extraction step 104, viable cells' walls are disrupted by such an additive, viable cells die, and thus ATP is extracted (128). On the other hand, ATPase loses its activity (129) due to the effect of enzyme denaturation by the additive, so as to preserve and protect ATP from degradation even after ATP is extracted from viable cells.
In the luminescent reaction of ATP with a luminescent reagent and luminescence measurement step 105, the thus extracted viable-cell-derived ATP is brought into contact with a luminescent reagent 108, so that a luminescent reaction 130 takes place. The thus generated luminescence is quantified using a luminescence measurement apparatus such as a luminometer. The luminescence level is proportional to the viable-cell-derived ATP count, and the ATP count is proportional to the viable cell count. Hence, the viable cell count can be estimated from the luminescence level. In addition, these 3 steps (the extracellular ATP removal step 103 (for removing ATP outside of viable cells), the intracellular ATP extraction step 104, and the ATP luminescent reaction•measurement step 105) proceed irreversibly.
The intracellular ATP level per viable cell is about 1.5×10−18 mol/CFU (0.001 fmol/CFU=1 amol/CFU) in terms of 1 bacterial CFU (Non-patent Document 1). The sensitivity of ATP measurement using a general ATP luminescence method ranges from 1×10−15 to 1×10−16 mol (1 to 0.1 fmol). The present inventors have achieved the sensitivity of ATP measurement of 1.0×10−18 mol and attempted the measurement of one viable cell. However, when such ATP luminescence method is used in the pharmaceutical manufacturing field and particularly for the control of pharmaceutical water, microorganisms contained in extremely pure water, such as purified water and water for injection, or in other words, water, in which almost no nutrient is present, must be detected. In such a case, conventional techniques still have a problem that one viable cell cannot be measured.